Substituted aminothienopyridines, pharmaceutical composition and use

ABSTRACT

This application relates to compounds of the formula ##STR1## wherein R 1 , R 2 , R 3 , m and n are as defined in the specification which are useful as modulators of neurotransmitter function such as serotonergic and adrenergic, and as such are useful as antidepressants, anxiolytics, atypical antipsychotics, antiemetics, and for the treatment of personality disorders such as obsessive compulsive disorders. Certain of the compounds are also useful as glycine partial agonists.

This is a continuation, of application Ser. No. 08/451,585, filed May26, 1995, now abandoned which is a divisional of Ser. No. 07/132,731,filed Oct. 6, 1993, now U.S. Pat. No. 5,519,032, which is a divisionalof Ser. No. 07/917,247, filed Jul. 20, 1992, now U.S. Pat. No.5,252,581, which are herein incorporated by reference.

The present invention relates to compounds of the formula, ##STR2##where R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃ -C₆)alkynyl,aryl(C₁ -C₆)alkyl, (C₁ -C₆)alkylcarbonyl, formyl, (C₁-C₆)alkoxycarbonyl, aryl(C₁ -C₆)alkoxycarbonyl, aryl(C₁-C₆)alkoxycarbonylamino(C₁ -₁₈)alkylcarbonyl, (C₁-C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl, (C₁ -C₆)alkylamino(C₁-C₆)alkylcarbonyl, amino(C₁ -C₁₈)alkylcarbonyl, (C₁ -C₆)dialkylamino(C₁-C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkyl,or (C₁ -C₆)dialkylamino(C₁ -C₆)alkyl;

R² is hydrogen, (C₁ -C₆)alkyl or ##STR3## with the proviso that R¹ andR² are not concurrently hydrogen; and

R³ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl;

where X is hydrogen, (C₁ -C₆)alkyl, halo, (C₁ -C₆)alkoxy or nitro;

n is 0, 1, 2 or 3;

m is 0, 1, 2 or 3; with the proviso that the sum of m and n is always 3;and

p is 0 or 1; and

pharmaceutically acceptable addition salts thereof and optical orgeometrical isomers or racemic mixtures thereof;

which compounds are useful as modulators of neurotransmitter functionsuch as serotonergic and adrenergic, and as such are useful asantidepressants, anxiolytics, atypical antipsychotics, antiemetics, andfor the treatment of personality disorders such as obsessive compulsivedisorders. Certain of the compounds are also useful as glycine partialagonists. This invention also relates to pharmaceutical compositionscontaining these compounds, methods of their use and a process formaking these compounds.

Unless otherwise stated or indicated, the following definitions shallapply throughout the specification and the appended claims.

The term (C₁ -C₆)alkyl and (C₁ -C₁₈)alkyl shall mean a straight orbranched alkyl group, for example, methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, sec-butyl, t-butyl and straight- and branched-chainpentyl and hexyl, and, in the case of (C₁ -C₁₈)alkyl optionallysubstituted by hydroxymethyl or trifluoromethyl.

The term halo(halogen) shall mean fluorine, chlorine, bromine or iodine.

The term aryl shall mean a phenyl group substituted with 0, 1 or 2substituents each of which being independently (C₁ -C₆)alkyl, (C₁-C₆)alkoxy, halogen, nitro or trifluoromethyl.

Throughout the specification and the appended claims, a given chemicalformula or name shall encompass all stereo, optical and tautomericisomers where such isomers exist.

In one class of compounds of this invention are compounds of the formula##STR4## wherein R¹, R² and R³ are as defined above.

In one preferred embodiment of this class are compounds of the formula##STR5## wherein R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃-C₆)alkynyl, aryl(C₁ -C₆)alkyl, (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkoxycarbonyl, amino(C₁ -C₁₈)alkylcarbonyl,(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl or (C₁ -C₆)alkylamino(C₁-C₆)alkyl; and

R³ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl;

where X is hydrogen, (C₁ -C₆)alkyl, halo or nitro; and p is 0 or 1.

More preferably, in this embodiment

R¹ is hydrogen, C₁ -C₆)alkyl or (C₁ -C₆)alkylcarbonyl;

R³ is hydrogen or (C₁ -C₆)alkoxycarbonyl;

X is hydrogen; and

p is 0.

Most preferably, the compounds have the formula ##STR6## where R¹ ishydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkylcarbonyl; and R³ is hydrogen.

In another embodiment of this class are compounds of the formula##STR7## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, amino(C₁ -C₁₈)alkylcarbonyl, (C₁ -C₆)alkylamino(C₁-C₆)alkylcarbonyl or (C₁ -C₆)dialkylamino(C₁ -C₆)alkylcarbonyl; and

R³ is hydrogen.

Most preferably,

R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁)alkylcarbonyl oramino(C₁)alkylcarbonyl.

In another class of compounds of this invention are compounds of theformula ##STR8## wherein R¹, R² and R³ are as defined above.

In a preferred embodiment of this class are compounds of the formula##STR9## wherein R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃-C₆)alkynyl, aryl(C₁ -C₆)alkyl, (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkoxycarbonyl, amino(C₁ -C₁₈)alkylcarbonyl,(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl or (C₁ -C₆)alkylamino(C₁-C₆)alkyl; and R³ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl;

where X is hydrogen, (C₁ -C₆)alkyl, halo or nitro; and p is 0 or 1.

More preferably, in this embodiment R¹ is hydrogen, (C₁ -C₆) or (C₁-C₆)alkylcarbonyl; R³ is hydrogen or (C₁ -C₆)alkoxycarbonyl; X ishydrogen; and p is 0.

Most preferably, the compounds have the formula ##STR10## where R¹ ishydrogen, (C₁ -C₆) or (C₁ -C₆)alkylcarbonyl; and R³ is hydrogen.

In another embodiment of this class are compounds of the formula##STR11## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl or amino(C₁ -C₁₈)alkylcarbonyl and

R³ is hydrogen.

Most preferably,

R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁)alkylcarbonyl oramino(C₁)alkylcarbonyl.

In another class of compounds of this invention are compounds of theformula ##STR12## wherein R¹, R² and R³ are as defined above.

In a preferred embodiment of this class are compounds of the formula##STR13## wherein R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃-C₆)alkynyl, aryl(C₁ -C₆)alkyl, (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkoxycarbonyl, amino(C₁ -C₁₈)alkylcarbonyl,(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl or (C₁ -C₆)alkylamino(C₁-C₆)alkyl; and

R³ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl ;

where X is hydrogen, (C₁ -C₆)alkyl, halo or nitro; and p is 0 or 1.

More preferably in this embodiment R¹ is hydrogen, (C₁ -C₆)alkyl or (C₁-C₆)alkylcarbonyl; R³ is hydrogen or (C₁ -C₆)alkoxycarbonyl; X ishydrogen; and p is 0.

Most preferably, the compounds have the formula ##STR14## where R¹ ishydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkylcarbonyl; and R³ is hydrogen.

In another embodiment of this class are compounds of the formula##STR15## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl or amino(C₁ -C₁₈)alkylcarbonyl and

R³ is hydrogen.

Most preferably, R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁)alkylcarbonyl oramino(C₁)alkylcarbonyl.

In yet another class of compounds of this invention are compounds of theformula ##STR16## wherein R¹, R² and R³ are as defined above.

In a preferred embodiment of this call are compounds of the formula##STR17## wherein R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃-C₆)alkynyl, aryl(C₁ -C₆)alkyl, (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkoxycarbonyl, amino(C₁ -C₁₈)alkylcarbonyl,(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl or (C₁ -C₆)alkylamino(C₁-C₆)alkyl; and

R³ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl;

where X is hydrogen, (C₁ -C₆)alkyl, halo or nitro; and p is 0 or 1.

More preferably in the embodiment

R¹ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkylcarbonyl;

R³ is hydrogen or (C₁ -C₆)alkoxycarbonyl;

X is hydrogen; and

p is 0.

Most preferably, the compounds have the formula ##STR18## where R¹ ishydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkylcarbonyl; and R³ is hydrogen.

In another embodiment of this class are compounds of the formula##STR19## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl or amino(C₁ -C₁₈)alkylcarbonyl and

R³ is hydrogen.

Most preferably, R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁)alkylcarbonyl oramino(C₁)alkylcarbonyl.

Nonlimiting examples of compounds of this invention include:

3-(4-pyridinylamino)thieno 2,3-b!pyridine;

3-(propyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(methyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(ethyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(butyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(4-pyridinylamino)thieno 3,2-c!pyridine;

3(propyl-4-pyridinylamino)thieno 3,2-c!pyridine;

3-(methyl-4-pyridinylamino)thieno 3,2-c!pyridine;

3-(ethyl-4-pyridinylamino)thieno 3,2-c!pyridine;

3-(butyl-4-pyridinylamino)thieno 3,2-c!pyridine;

3-(4-pyridinylamino)thieno 3,2-b!pyridine;

3-(propyl-4-pyridinylamino)thieno 3,2-b!pyridine;

3-(methyl-4-pyridinylamino)thieno 3,2-b!pyridine;

3-(ethyl-4-pyridinylamino)thieno 3,2-b!pyridine;

3-(butyl-4-pyridinylamino)thieno 3,2-b!pyridine;

3-(4-pyridinylamino)thieno 2,3-c!pyridine;

3-(methyl-4-pyridinylamino)thieno 2,3-c!pyridine;

3-(ethyl-4-pyridinylamino)thieno 2,3-c!pyridine;

3-(propyl-4-pyridinylamino)thieno 2,3-c!pyridine;

3-(butyl-4-pyridinylamino)thieno 2,3-c!pyridine;

3-(acetyl-4-pyridinylamino)thieno 2,3-c!pyridine;

3-(acetyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(propionyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(acetyl-4-pyridinylamino)thieno 3,2-c!pyridine;

3-(propionyl-4-pyridinylamino)thieno 3,2-c!pyridine;

3-(acetyl-4-pyridinylamino)thieno 3,2-b!pyridine;

3-(propionyl-4-pyridinylamino)thieno 2,3-b!pyridine;

3-(propionyl-4-pyridinylamino)thieno 2,3-c!pyridine;

2-amino-N-(thieno 2,3-b!pyridin-3-yl)acetamide;

2-amino-N-(thieno 3,2-c!pyridin-3-yl)acetamide;

2-amino-N-(thieno 3,2-b!pyridin-3-yl)acetamide;

2-amino-N-(thieno 2,3-c!pyridin-3-yl)acetamide;

t-butyl 2-(thieno 2,3-b!pyridin-3-ylamino)-2-oxoethyl!carbamate;

t-butyl 2-(thieno 3,2-c!pyridin-3-ylamino)-2-oxoethyl!carbamate;

t-butyl 2-(thieno 3,2-b!pyridin-3-ylamino)-2-oxoethyl!carbamate;

t-butyl 2-(thieno 2,3-c!pyridin-3-ylamino)-2-oxoethyl!carbamate;

The compounds of the invention are prepared by one or more of thesynthetic routes described below.

Throughout the description of the synthetic schemes, the notations R¹,R³, X, m, n and p have the respective meanings given above unlessotherwise stated or indicated and other notations have the respectivemeanings defined in their first appearances.

More particularly, as shown in Reaction Scheme A, an aminothienopyridineof Formula XVIII is reacted with a halopyridine of Formula XIX or thecompound of Formula XX wherein R⁴ is hydrogen, (C₁ -C₆)alkyl orhydroxy(C₁ -C₆)alkyl, R⁵ is hydrogen or (C₁ -C₆)alkyl, R⁶ is hydrogen or(C₁ -C₆)alkyl, R⁷ is (C₁ -C₆)alkyl or aryl(C₁ -C₆)alkyl and k and q areindependently 0 to 5 with the proviso that the sum of k and q is notgreater than 5, to obtain the N-substituted compound of Formula XXI orXXII, respectively.

When the product is the compound of Formula XXI on Scheme A, thereaction is generally carried out in an ethereal solvent such asbis(2-methoxyethyl)ether, diethyl ether, dimethoxyethane, dioxane ortetrahydrofuran or a polar aprotic solvent such as dimethylformamide,dirnethylacetamide, N-methyl-2-pyrrolidone, hexamethylphosphoramide ordimethyl sulfoxide or polar solvent such as ethanol or isopropanol at atemperature of between about 0° C. and 200° C., preferably between about20° C. and 150° C., most preferably between about 50° C. and 100° C.

The compound of Formula XXI is then allowed to react with a halide orsulfate of the formula R⁸ hal where R⁸ is (C₁ -C₆)alkyl, (C₃-C₆)alkenyl, (C₃ -C₆)alkynyl or aryl(C₁ -C₆)alkyl, or (R⁹ O)₂ SO₂ whereR⁹ is (C₁ -C₆)alkyl, at a temperature of from about -10° C. to about 80°C., preferably from about 0° C. to about 25° C. to obtain the compoundof Formula III, VII, XI or XV where R¹ corresponds to R⁸ or R⁹ above.

In the case where the product is the compound of Formula XXII thereaction is carried out in the presence of a condensing agent such asdicyclohexylcarbodiimide (DCC.). Typically, the reaction is carried outin a suitable solvent such as an aprotic organic solvent, for example,dichloromethane, dioxane or tetrahydrofuran at a temperature of fromabout 0° C. to about 100° C., preferably from about 15° C. to about 80°C., most preferably from about 20° C. to about 50° C.

The compound of Formula XXII is deprotected by means known in the art.In the case where R⁷ is phenylmethyl, the compound of Formula XXII isdeprotected, for example, by treatment with hydrogen in the presence ofa catalyst such as ##STR20## palladium on carbon to yield the aminocompound of Formula V, IX, XIII or XVII. The reaction is typicallycarried out in a polar solvent such as methanol or ethanol at about 10°C. to 50° C., preferably from about 15° C. to about 30° C.

When R⁷ of Formula XXII is t-butyl, the compound is deprotected by meansknown in the art, for example, by treatment with acid such ashydrochloric acid, hydrobromic acid or trifluoroacetic acid in anorganic solvent such as ethyl acetate, tetrahydrofuran, methanol,chloroform and the like, or neat, at from about -50° C. to about 100° C.Preferably, the reaction is carried out in the presence of hydrochloricacid in methanol at about -15° C. to about 30° C.

The aminothienopyridine starting materials are prepared according tomethods known in the art, for example, reaction of the appropriatecyanohalopyridine with methylthioglycolate (J. Het. Chem., 24 85 (1987),J. Het. Chem., 7 373 (1970)) followed by removal of the 2-ester group asoutlined in Reaction Scheme B for 3-aminothieno 2,3-b!pyridine and3-aminothieno 2,3-c!pyridine. The starting pyridine compound3-chloro-4-cyanopyridine can be prepared from 4-cyanopyridine N-oxide bymeans known in the art (J. Het. Chem., 15 683 (1978)). ##STR21##

The compounds of Formula III, V, VII, IX, XI, XIII, XV and XVII of thepresent invention are useful as modulators of neurotransmitter functionsuch as serotonergic and adrenergic and, as such, are useful asantidepressants, anxiolytics, atypical antipsychotics, antiemetics, andfor the treatment of personality disorders such as obsessive compulsivedisorders. Certains of the compounds are also useful as glycine partialagonists.

³ H!-8-Hydroxy-2-(di-n-propylamino)-tetralin ( ³ H!DPAT) Binding toSerotonin (5HT_(1A)) Receptors

Purpose:

The purpose of this assay is to determine the affinity of test compoundsfor the 5HT_(1A) receptor in brain. It is believed to be useful forpredicting compounds with serotonergic properties with potential utilityas novel anxiolytics (1-4), atypical antipsychotics or useful in thetreatment of personality disorders such as obsessive compulsivedisorder.

Introduction

The existence of two populations of 5HT receptors in rat brain was shownby differential sensitivity to spiroperidol (5). Thespiroperidol-sensitive receptors were designated as the 5HT_(1A) subtypeand the insensitive receptors were referred to as the 5HT_(1B) subtype(6). Other 5HT binding sites (5HT_(1C), 5HT_(1D) and 5HT₃) havesubsequently been identified in various species, based on differentialsensitivity to 5HT antagonists (7). A significant advance in theclassification of 5HT receptors came with the identification of aselective ligand for the ⁵ HT_(1A) receptor, ³ H!DPAT (8). These authorsreported that ³ H!DPAT labeled an autoreceptor. Lesion studies suggestthat ³ H!DPAT labeled receptors are not terminal autoreceptors, but maybe somatodendritic autoreceptors (9)). Although DPAT decreases thefiring rate in the Raphe nucleus and inhibits 5HT release, the actuallocation and function is somewhat controversial (2). These studies andthe sensitivity of ³ H!DPAT binding to guanine nucleotides and effectson adenylate cyclase suggest that DPAT acts as an agonist at the5HT_(1A) receptor (10).

Procedure I:

A. Reagents

Tris Buffers, pH7.7

(a) 57.2 g Tris HCl

16.2 g Tris base

Bring volume to 1 liter with distilled water (0.5M Tris buffer, pH 7.7).

(b) Make a 1:10 dilution in deionized H₂ O (0.05M Tris buffer, pH 7.7).

(c) 0.05M Tris buffer, pH 7.7 containing 10 μM pargyline, 4 mM CaCl₂ and0.1% ascorbic acid.

0.49 mg pargyline•HCl

111 mg CaCl₂

250 ascorbic acid

Bring to 250 ml with 0.05M Tris buffer, pH

7.7 (reagent 1b)

2. 8-Hydroxy ³ H!-DPAT (2-(N,N-Di 2,3(n)-³H!propylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene) (160-206Ci/mmol) was obtained from Amersham.

For IC₅₀ determinations: a 10 nM stock solution is made up and 50 μladded to each tube (final concentration=0.5 nM).

3. Serotonin creatinine sulfate. 0.5 mM stock solution is made up in0.01N HCl and 20 μl added to 3 tubes for determination of nonspecificbinding (final concentration=10 μM).

4. Test Compounds. For most assays, a 1 mM stock solution is made up ina suitable solvent and serially diluted, such that the finalconcentration in the assay ranges from 2×10⁵ to 2×10⁻⁸ M. Sevenconcentrations are used for each assay. Higher or lower concentrationsmay be used based on the potency of the drug.

B. Tissue Preparation

Male Wistar rats are sacrificed by decapitation. Hippocampi are removed,weighed and homogenized in 20 volumes of 0.05M Tris buffer, pH 7.7. Thehomogenate is centrifuged at 48,000 g for 10 minutes and the supernatantis discarded. The pellet is resuspended in an equal volume of 0.05M Trisbuffer, incubated at 37° C. for 10 minutes and recentrifuged at 48,000 gfor 10 minutes. The final membrane pellet is resuspended in 0.05M Trisbuffer containing 4 mM CaCl₂, 0.1% ascorbic acid and 10 μM pargyline.

C. Assay

800 μl Tissue

130 μl 0.05M Tris+CaCl₂ +pargyline+ascorbic acid

20 μl vehicle/5HT/drug

50 μl ³ H!DPAT

Tubes are incubated for 15 minutes at 25° C. The assay is stopped byvacuum filtration through Whatman GF/B filters which are then washed 2times with 5 ml of ice-cold 0.05M Tris buffer. The filters are thenplaced into scintillation vials with 10 ml of Liquiscint scintillationcocktail and counted.

Calculation

Specific binding is defined as the difference between total binding andbinding in the presence of 10 μM 5HT. IC₅₀ values are calculated fromthe percent specific binding at each drug concentration.

Procedure II:

A. Reagents

1. Tris Buffers, pH 7.7

(a) 57.2 g Tris HCl

16.2 g Tris base

Bring to 1 liter with distilled water (0.5M Tris buffer, pH 7.7).

(b) Make a 1:10 dilution in distilled H₂ O (0.05M Tris buffer,

pH 7.7 at 25° C.).

(c) 0.05M Tris buffer, pH 7.7 containing 10 μM

pargyline, 4 mM CaCl₂ and 0.1% ascorbic acid.

0.49 mg pargyline•HCl

110.99 mg CaCl₂

250 ascorbic acid

Bring to 250 ml with 0.05M Tris buffer, pH

7.7 (reagent 1b).

2. 8-Hydroxy ³ H!-DPAT (2-N,N-Di 2,3(n)-³H!propylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene)! (160-206Ci/mmol) is obtained from Amersham.

For IC₅₀ determinations: ³ H-DPAT is made up to a concentration of 3.3nM in the Tris Buffer (1c) such that when 150 μl is added to each tube afinal concentration of 0.5 nM is attained in the 1 ml assay.

3. Serotonin creatinine sulfate is obtained from the Sigma ChemicalCompany. Serotonin creatinine sulfate is made up to a concentration of100 μM in Tris buffer (1c). One hundred μl is added to each of 3 tubesfor the determination of nonspecific binding (this yields a finalconcentration of 10 μM in the 1 ml assay).

4. Test Compounds. For most assays, a 100 μM stock solution is made upin a suitable solvent and serially diluted with Tris buffer (1c) suchthat when 100 μl of drug is combined with the total 1 ml assay, a finalconcentration ranging from 10⁻⁵ to 10⁻⁸ M is attained.Characteristically seven concentrations are studied for each assay;however, higher or lower concentrations may be used, depending on thepotency of the drug.

B. Tissue Preparation

Male Wistar rats are decapitated, the hippocampi are removed andhomogenized in 20 volumes of ice cold 0.05M Tris buffer, pH 7.7 (1b).The homogenate is centrifuged at 48,000 g for 10 minutes at 4° C. Theresulting pellet is rehomogenized in fresh Tris buffer (1b), incubatedat 37° C. for 10 minutes and recentrifuged at 48,0000 g for 10 minutes.The final membrane pellet is resuspended in 0.05M Tris buffer (1c)containing 4 mM CaCl₂, 0.1% ascorbic acid and 10 μM pargyline. Specificbinding is approximately 90% of total bound ligand.

C. Assay

750 μl Tissue

150 μl ³ H!DPAT

100 μl vehicle (for total binding) or 100 μM serotonin creatininesulfate (for nonspecific binding) or appropriate drug concentration

Tubes are incubated for 15 minutes at 25° C. The assay is stopped byvacuum filtration through Whatman GF/B filters which are then washed 2times with 5 ml of ice-cold 0.05M Tris buffer (1b). The filters are thenplaced into scintillation vials with 10 ml of Liquiscint scintillationcocktail and counted. Specific binding is defined as the differencebetween total binding in the absence or presence of 10 μM serotonincreatinine sulfate. IC₅₀ values are calculated from the percent specificbinding at each drug concentration.

The K_(D) value for ³ H! DPAT binding was found to be 1.3 nM byScatchard analysis of a receptor saturation experiment. The K_(i) valuemay then be calculated by the Cheng-Prusoff equation:

    K.sub.i =IC.sub.50 /1+L/K.sub.D

References:

1. Dourish C. T., Hutson, P. H. and Curzon, G.: Putative anxiolytics8-OH-DPAT, buspirone and TVX Q 7821 are agonists at 5 HT_(1A)autoreceptors in the raphe nucleus. TIPS 7: 212-214(1986).

2. Verge, D., Daval, G., Marcinkiewicz, M., Patey, A., El Mestikawy, H.Gozlan and Hamon, M.: Quantitative autoradiography of multiple 5-HT₁receptor subtypes in the brain of control or5,7,dihydroxytryptamine-treated rats. J. Neurosci. 6: 3474-3482 (1986).

3. Iversen, S. D.: 5HT and anxiety. Neuropharmacol. 23: 1553-1560(1984).

4. Traber J. and Glaser, T.:5HT_(1A) receptor-related anxiolytics. TIPS8: 432-437 (1987).

5. Pedigo, N. W., Yammamura, H. I. and Nelson, D. L.: Discrimination ofmultiple ³ H!5-hydroxytryptamine binding sites by the neurolepticspiperone in rat brain. J. Neurochem. 36: 220-226 (1981).

6. Middlemiss, D. N. and Fozard J. R.:8-Hydroxy-2-(di-n-propylamino)tetraline discriminates between subtypesof the 5HT₁ recognition site. Eur. J. Pharmacol. 90: 151-152(1983).

7. Peroutka, S. J.: Pharmacological differentiation and characterizationof 5-HT_(1A), 5-HT_(1B) and 5-HT_(1C) binding sites in rat frontalcortex. J. Neurochem. 47: 529-540 (1986).

8. Peroutka, S. J.: 5-Hydroxytryptamine receptor subtypes: molecular,biochemical and physiological characterization TINS 11: 496-500 (1988).

9. Gozlan, H., El Mestikawy, S., Pichat, L. Glowinsky, J. and Hamon, M.:Identification of presynaptic serotonin autoreceptors using a newligand: ³ H-PAT. Nature 305: 140-142 (1983).

10. Schlegel,.R. and Peroutka, S. J.: Nucleotide interactions with5-HT_(1A) binding sites directly labeled by ³H!-8-hydroxy-2-(di-n-propylamino)tetralin ( ³ H!-8-OH-DPAT). Biochem.Pharmacol. 35: 1943-1949 (1986).

11. Peroutka, S. J.: Selective interaction of novel anxiolytics with5-hydroxytryptamine_(1A) receptors. Biol. Psychiatry. 20: 971-979(1985).

³ H-Serotonin Uptake in Rat Whole Brain Synaptosomes

Purpose:

This assay is used as a biochemical screen for potential antidepressantswhich block serotonin (5HT) uptake, which may be useful for thetreatment of personality disorders such as obsessive compulsivedisorder.

Introduction:

Asberg and coworkers have suggested that subjects with serotonergichypofunction comprise a biochemical subgroup of depressed patients (1),while others (2) claim that altered serotonergic function determines themood changes associated with affective disorders. Although the role of5HT in the etiology of depression is not clear; it is true that a numberof antidepressant drugs block the 5HT uptake mechanism. In vitroreceptor binding assays have shown that ³ H!-imipramine labels 5HTuptakes sites (10). Trazodone and zimelidine are clinically effectiveantidepressants (3) with fairly selective effects on 5HT uptake (4,5).More recently, fluoxetine has been shown to be both a selective andpotent 5HT uptake inhibitor.

³ H!-5HT transport has been characterized in CNS tissue (6,7) and foundto be saturable, sodium- and temperature-dependent, inhibited byouabain, metabolic inhibitors, tryptamine analogs (8) and tricyclicantidepressants (tertiary amines>>secondary amines) (9). The latterfindings differentiate 5HT uptake from catecholamine uptake. ³ H!-5HTuptake can also be used as a marker for serotonin nerve terminals.

Procedure:

A. Animals: Male CR Wistar rats (100-125 g).

B. Reagents

1. Krebs-Henseleit Bicarbonate Buffer, pH 7.4 (KHBB): Make a 1 literbatch, containing the following salts.

    ______________________________________                    g/L     mM    ______________________________________    NaCl              6.92      118.4    KCl               0.35      4.7    MgSO.sub.4.7H.sub.2 O                      0.29      1.2    KH.sub.2 PO.sub.4 0.16      2.2    NaHCO.sub.3       2.10      24.9    CaCl.sub.2        0.14      1.3    Prior to use add:    Dextrose          2 mg/ml   11.1    Iproniazid phosphate                      0.30 mg/ml                                0.1    ______________________________________

Aerate for 60 min. with 95% O₂ /5% CO₂, check pH (7.4±0.1)

2. 0.32M Sucrose: 21.9 g of sucrose, bring to 200 ml.

3. Serotonin creatinine SO₄ is obtained from Sigma Chemical Co. A 0.1 mMstock solution is made up in 0.01N HCl . This is used to dilute thespecific activity of radiolabeled 5HT.

4. 5- 1,2-³ H(N)!-Hydroxytryptamine creatinine sulfate (Serotonin),specific activity 20-30 Ci/mmol is obtained from New England Nuclear.

The final desired concentration of ³ H-5HT in the assay is 50 nM. Thedilution factor is 0.8 Therefore, the KHBB is made up to contain 62.5 nM³ H!-5HT.

Add to 100 ml of KHBB.

    ______________________________________     A) 56.1 μl of 0.1 mM 5HT                      =         56.1 nM    *B) 0.64 nmole of .sup.3 H-5HT                      =          6.4 nM                                62.5 nM    ______________________________________     *Calculate volume added from specific activity of .sup.3 H5HT.

5. For most assays, a 1 mM solution of the test compound is made up insuitable solvent and serially diluted such that the final concentrationin the assay ranges from 2×10⁻⁸ to 2×10⁻⁵ M. Seven concentrations areused for each assay. Higher or lower concentrations may be useddepending on the potency of the compound.

C. Tissue Preparation

Male Wistar rats are decapitated and the brain rapidly removed. Wholebrain minus cerebella is weighed and homogenized in 9 volumes ofice-cold 0.32M sucrose using a Potter-Elvejhem homogenizer.Homogenization should be done with 4-5 up and down strokes at mediumspeeds to minimize synaptosome lysis. The homogenate is centrifuged at1000 g for 10 min. at 0°-4° C. The supernatant (S₁) is decanted and isused for uptake experiments.

D. Assay

800 μl KHBB+ ³ H!-5HT

20 μl Vehicle or appropriate drug concentration

200 μl Tissue suspension

Tubes are incubated at 37° C. under a 95% O₂ /5% CO₂ atmosphere for 5minutes. For each assay, 3 tubes are incubated with 20 μl of vehicle at0° C. in an ice bath. After incubation all tubes are immediatelycentrifuged at 4000 g for 10 minutes. The supernatant fluid is aspiratedand the pellets dissolved by adding 1 ml of solubilizer (TritonX-100+50% EtOH, 1:4 v/v). The tubes are vigorously vortexed, decantedinto scintillation vials, and counted in 10 ml of Liquiscintscintillation counting cocktail. Active uptake is the difference betweencpm at 37° C. and 0° C. The percent inhibition at each drugconcentration is the mean of three determinations. IC₅₀ values arederived from log-probit analysis.

References:

1. Asberg, M., Thoren, P., Traskman, L., Bertilsson, L., and Ringberger,V. "Serotonin depression:-A biochemical subgroup within the affectivedisorders. Science 191: 478-480 (1975).

2. DeMontigy, C. Enhancement of 5HT neurotransmission by antidepressanttreatments. J. Physiol. (Paris) 77: 455-461 (1980).

3. Feighner, J. P. Clinical efficacy of the newer antidepressants. J.Clin. Psychopharmacol. 1: 235-265 (1981).

4. Ogren, S. O., Ross, S. B., Hall, H., Holm, A. C. and Renyi, A. L. Thepharmacology of zimelidine: A 5HT selective reuptake inhibitor. ActaPsychiat. Scand. 290: 127-151 (1981).

5. Clements-Jewry, S., Robson, P. A. and Chidley, L. J. Biochemicalinvestigations into the mode of action of trazodone. Neuropharmacol. 19:1165-1173 (1980).

6. Ross, S. B. Neuronal transport of 5-hydroxytryptamine. Pharmacol. 21:123-131 (1980).

7. Shaskan, E. G. and Snyder, S. H. Kinetics of serotonin accumulationinto slices from rat brain: Relationship to catecholamine uptake. J.Pharmacol. Exp. Ther. 175: 404-418 (1970).

8. Horn, S. A. Structure activity relations for the inhibition of 5HTuptake into rat hypothalamic homogenates by serotonin and tryptamineanalogues. J. Neurochem. 21: 883-888 (1973).

9. Horn, A. S. and Trace, R. C. A. M. Structure-activity relations forthe inhibition of 5-hydroxytryptamine uptake by tricyclicantidepressants into synaptosomes from serotonergic neurones in ratbrain homogenates. Brit. J. Pharmacol. 51: 399-403 (1974).

10. Langer, S. Z., Moret, C., Raisman, R., Dubocovich, M. L. and BrileyM. High affinity ³ H!imipramine binding in rat hypothalamus: Associationwith uptake of serotonin but not norepinephrine. Science 210: 1133-1135(1980).

Results of the two assay methods described above are presented in TableI for representative compounds of this invention.

                                      TABLE I    __________________________________________________________________________                    Inhibition of Biogenic Amine                    Reuptake IC.sub.50 (μM)                                          5HT.sub.3 Receptor                    .sup.3 H-Serotonin                          .sup.3 H-Norepinephrine                                   .sup.3 H-Dopamine                                          Binding Assay    Compound        (WB)  (WB)     (Str)  IC.sub.50 (μM)    __________________________________________________________________________    3-(Propyl-4-pyridinylamino)-                    0.071 0.71     0.30   4.6    thieno 2,3-b!pyridine hydrochloride    3-(Propyl-4-pyridinylamino)-                    0.54    thieno 2,3-c!pyridine    Clomipramine    0.033 0.36     31.5   >10    __________________________________________________________________________     wb = whole brain;     str. = striatum

³ H!Glycine Binding

Purpose:

This assay is used to assess the affinity of compounds for the glycinebinding site associated with the N-methyl-D-aspartate (NMDA) receptorcomplex using ³ H!glycine as the radioligand.

Introduction:

The amino acid glycine modulates and may be a requirement for theactivation of the excitatory amino acid receptors of the NMDA subtype(1). Glycine has been shown in vitro to potentiate the effects of1-glutamate or NMDA on the stimulation of ³ H!TCP binding (2,3,4) and ³H!norepinephrine release (5), and in vivo to act as a positive modulatorof the glutamate-activated cGMP response in the cerebellum (6,7). Theactivation of NMDA receptors requiring the presence of glycine isnecessary for the induction of long-term potentiation (LTP), a type ofsynaptic plasticity which may be fundamental to learning processes (8).A ³ H!glycine binding site in the brain has been identified andcharacterized as a strychnine-insensitive site associated with the NMDAreceptor complex (10, 11, 12). Autoradiographic studies have shown asimilar distribution of ³ H!glycine and ³ H!TCP (NMDA ion channelradioligand) binding sites (13, 14). Compounds which interact with theglycine site offer a novel mechanism of action for intervention withNMDA receptor function.

Procedure:

A. Reagents

1. Buffer A: 0.5M Tris maleate, pH 7.4

59.3 g Tris maleate bring to 0.51 with distilled water Adjust pH to 7.4with 0.5M Tris base.

2. Buffer B: 50 mM Tris maleate, pH 7.4

Dilute Buffer A 1:10 with distilled water; adjust pH with 50 mM Trismaleate (acid) or 50 mM Tris base.

3. Glycine, 5×10⁻² M.

Dissolve 3.755 mg of glycine (Sigma G7126) with 1.0 ml distilled water.Aliquots of 20 μl to the assay tube will give a final concentration of10⁻³ M.

4. ³ H!Glycine is obtained from New England Nuclear, specific activity45-50 Ci/mmole. For IC₅₀ determinations, a 200 nM stock solution is madewith distilled water. Aliquots of 50 μl are added to yield a final assayconcentration of 10 nM.

5. Test compounds. A stock solution of 5 mM is made with a suitablesolvent and serially diluted, such that the final concentration in theassay ranges from 10⁻⁴ to 10⁻⁷ M. Higher or lower concentrations may beused, depending on the potency of the compound.

6. Triton-X 100, 10% (v/v) (National Diagnostics, EC-606). A stocksolution of Triton-X 100, 10% can be prepared and stored in therefrigerator. Dilute 1.0 ml of Triton-X 100 to 10.0 ml with distilledwater. On the day of the assay, the tissue homogenate (1:15 dilution) ispreincubated with an aliquot of the 10% solution to give a finalconcentration of 0.04% (v/v).

B. Tissue Preparation

Cortices of male Wistar rats are dissected over ice and homogenized inice-cold 0.32M sucrose (1:15 W/V) for 30 seconds with a Tissumizersetting at 70. Three cortices are pooled for one preparation. Thehomogenate is centrifuged at 1,000 g for 10 min (SS34, 3,000 rpm, 4°C.). The supernatant is centrifuged at 20,000 g (SS34, 12,000 rpm, 4°C.) for 20 minutes. The pellet is resuspended in 15 volumes of ice-colddistilled water (Tissumizer setting 70, 15 sec) and spun at 7,600 g(SS34, 8,000 rpm, 4° C.) for 20 minutes. The supernatant is saved. Theupper buffy layer of the pellet is swirled off and added to thesupernatant. The supernatant is centrifuged at 48,000 g (SS34, 20,000rpm, 4° C.) for 20 minutes. The pellet is resuspended with 15 volumes ofcold distilled water and centrifuged. The supernatant is discarded andthe pellet is stored at -70° C.

On the day of the assay, the pellet is resuspended in 15 volumesice-cold 50 mM Tris maleate, pH 7.4. The homogenate is preincubated withTriton-X in a final concentration of 0.04% (v/v) for 30 minutes at 37°C. with agitation. The suspension is centrifuged at 48,000 g (SS34,20,000 rpm, 4° C.) for 20 minutes. The pellet is washed an additional 3times by resuspension with cold buffer and centrifugation. The finalpellet is resuspended in a volume 25 times the original wet weight.

C. Assay

1. Prepare assay tubes in triplicate.

    ______________________________________    380 μl  Distilled water    50 μl   Buffer A, 0.5M Tris maleate, pH 7.4    20 μl   Glycine, 10.sup.-3 M final concentration,               or distilled water or appropriate               concentration of inhibitor    50 μl    .sup.3 H!Glycine, final concentration 10 nM    500 μl  Tissue homogenate    1000 μl Final volume    ______________________________________

2. Following the addition of the tissue, the tubes are incubated for 20minutes in an ice-bath at 0°-4° C. The binding is terminated bycentrifugation (HS4, 7,000 rpm, 4° C.) for 20 minutes. The tubes arereturned to ice. The supernatant is aspirated and discarded. The pelletis rinsed carefully twice with 1 ml ice-cold buffer, avoiding disruptionof the pellet, and transferred to scintillation vials by vortexing thepellet with 2 ml of scintillation fluid, rinsing the tubes twice with 2ml and then adding an additional 4 ml of scintillation fluid.

3. Specific binding is determined from the difference of binding in theabsence or presence of 10⁻³ M glycine and is typically 60-70% of totalbinding. IC₅₀ values for the competing compound are calculated bylog-probit analysis of the data.

3 References:

1. Thomson, A. M. Glycine modulation of the NMDA receptor/channelcomplex. Trends in Neuroscience 12: 349-353, 1989.

2. Snell, L. D., Morter, R. S. and Johnson, K. M. Glycine potentiatesN-methyl-D-aspartate-induced ³ H!TCP binding to rat cortical membranes.Neurosci. Lett. 83: 313-317, 1987.

3. Snell, L. D., Morter, R. S. and Johnson, K. M. Structuralrequirements for activation of the glycine receptor that modulates theN-methyl-D-aspartate operated ion channel. Eur. J. Pharmacol. 156:105-110, 1988.

4. Bonhaus, D. W., Yeh, G.-C., Skaryak, L. and McNamara, J. O. Glycineregulation of the N-methyl-D-aspartate receptor-gated ion channel inhippocampal membranes Mol. Pharmacol. 36: 273-279, 1989.

5. Ransom. R. Q. and Deschenes, N. L. NMDA-induced hippocampal ³H!norepinephrine release is modulated by glycine. Eur. J. Pharmacol.156: 149-155, 1988.

6. Danysz, W., Wroblewski, J. T., Brooker, G and Costa, E. Modulation ofglutamate receptors by phencyclidine and glycine in the rat cerebellum:cGMP increase in vivo. Brain Res. 479: 270-276, 1989.

7. Rao, T. S., Cler, J. A., Emmett, M. R., Mick, S. J., Iyengar, S. andWood, P. L. Glycine, glycinamide and D-serine act as positive modulatorsof signal transduction at the N-methyl-D-aspartate (NMDA) receptor invivo: differential effects on mouse cerebellar cyclic guanosinemonophosphate levels. Neuropharmacol. 29: 1075-1080, 1990.

8. Oliver, M. W., Kessler, M., Larson, J., Schottler, F. and Lynch, G.Glycine site associated with the NMDA receptor modulates long-termpotentiation. Synapse 5: 265-270, 1990.

9. Kishimoto, J., Simon, J. R. and Aprison, M. H. Determination of theequilibrium dissociation constants and number of glycine binding sitesin several areas of the rat central nervous system, using asodium-independent system. J. Neurochem. 37: 1015-1024, 1981.

10. Kessler, M., Terramani, T., Lynch, B. and Baudry, M. A glycine siteassociated with N-methyl-D-aspartic acid receptors: characterization andidentification of a new class of antagonists. J. Neurochem. 52:1319-1328, 1989.

11. Monahan, J. B., Corpus, V. M., Hood, W. F., Thomas, J. W. andCompton, R. P. Characterization of a ³ H!glycine recognition site as amodulatory site of the N-methyl-D-aspartate receptor complex. J.Neurochem. 53: 370-375, 1989.

12. Cotman, C. W., Monaghan, D. T., Ottersen, O. P. and Storm-Mathsen,J. Anatomical organization of excitatory amino acid receptors and theirpathways. Trends in Neuroscience 10: 273-280, 1987.

13. Jansen, K. L. R., Dragunovi, M. and Faull, R. L. M. ³ H!Glycinebinding sites, NMDA and PCP receptors have similar distributions in thehuman hippocampus: an autoradiographic study. Brain Res. 482: 174-178,1989.

ENHANCEMENT OF ³ H!TCP BINDING

Glutamate is considered to be a major excitatory neurotransmitter in thecentral nervous system. In addition, glutamate has been postulated asbeing involved in a number of pathological conditions such as neuronaldamage and loss due to ischemic stress (e.g. stroke), and inneurodegenerative disorders including Huntington's disease, amyotrophiclateral sclerosis, neurolathyrism, Alzheimer's disease and others (1,2).A central dopaminergic-glutamatergic balance was also suggested asimportant for both akinetic motor disorders (e.g. Parkinson's disease)and psychosis (e.g. schizophrenia)(3).

Postsynaptic effects of glutamate are mediated by a variety of glutamatereceptor subtypes, which are classified as N-methyl-D-aspartate (NMDA)and non-NMDA (quisqualate, kainate) receptor subtypes. Of the glutamatereceptor subtypes, the NMDA receptor has been extensively investigated.The receptor is composed of an agonist binding site (the NMDA site), anda cation channel with binding sites for magnesium and other ligandsincluding PCP, TCP and dextromethorphan. A number of modulatory sitesassociated with the NMDA receptor have been identified, includingbinding sites for zinc, polyamines, and glycine(2). The glycine site mayprovide a therapeutic target for treatment of various types of cognitiveimpairments including Alzheimer's disease(4).

The glycine modulatory site (glycine B site) is insensitive tostrychnine, whereas a strychnine sensitive glycine binding siteassociated with spinal cord neurons has been designated as the glycine Asite. In extensively washed preparations of rat cortical membranes, NMDAincreases the specific binding of ³ H!TCP in a concentration dependentmanner (EC₅₀ =3.1 μM) and addition of glycine (1 μM) potentiates themaximal effect of NMDA by a factor of 1.7(5). This preparation may beused to evaluate the effect of compounds at the NMDA associatedstrychnine-insensitive glycine modulatory site. Compounds can becharacterized as glycine-like agonists (compounds producing an effectequivalent to the maximal effect of glycine) or glycine partial agonists(compounds producing less than the maximal effect of glycine ) at thissite. The prototypical glycine partial agonist is D-cycloserine(4).

Procedure(5):

Crude synaptosomal homogenates are prepared from cortical tissueobtained from male Sprague Dawley rats immediately after sacrifice orthat have been frozen at -60° C. for not more than one month. Tissue ishomogenized by Polytron (Brinkmann, setting 7, 60 s) in ice-cold 0.32Msucrose and centrifuged for 20 minutes at 1000 g. The resultingsupernatant is decanted and recentrifuged at 17,500 g. The resultingpellet is then resuspended in 50 vols. of ice-cold distilled water andlysed at 37° C. for 30 minutes followed by centrifugation at 36,000 gfor 20 minutes. The resulting pellet is carried through a second lysingand then washed by resuspension in 50 vols. of 10 mM HEPES: Na HEPESbuffer (pH 7.5 at 4° C.). The homogenate is centrifuged again (36,000 g;20 minutes), resuspended in 30 vols. of HEPES buffer and frozen at -60°C. until used for binding experiments. On the day binding is performed,the homogenate is thawed and washed three times with 30 vols. of bufferbefore use. There are no appreciable differences in the binding inhomogenates obtained from fresh compared to frozen tissue.

All binding studies are performed by incubating homogenates(approximately 0.2 mg protein per assay tube) with 2.5 nM ³ H!TCP (40Ci/mmol; New England Nuclear, Boston, Mass.) for 120 minutes at 25° C.in a final volume of 1 ml of 10 mM HEPES buffer (pH 7.5). Non-specificbinding is determined in the presence of 100 μM PCP. The assay tubeswere prepared in triplicate as follows:

    ______________________________________    360 μl           distilled water    50 μl           0.1M HEPES buffer, pH 7.5    20 μl           L-glutamic acid, 5 × 10.sup.-6 M (Final concentration =           10.sup.-7 M)    20 μl           glycine, final concentration 10.sup.-8 to 10.sup.-3 M, or           compound, final concentration 10.sup.-8 to 10.sup.-3 M,           or distilled water, or PCP, final concentration 100 μM    50 μl            .sup.3 H!TCP    500 μl           Tissue homogenate    1,000 μl           Final Volume    ______________________________________

The binding reaction is terminated by vacuum filtration on GF/C glassfiber filters which are presoaked for 20-30 minutes in 0.05%polyethyleneimine (Sigma) in order to reduce binding to the filters.Filtration is followed by 2 washes with 4 ml of ice-cold buffer and theretained radioactivity is measured by liquid scintillation spectrometry.Protein concentration is measured by the method of Bradford (6).

Initial experiments revealed that the amount of ³ H!TCP binding measuredvaries somewhat with each membrane preparation. Therefore, in eachexperiment, the specific binding of ³ H!TCP in the absence of anyadditional drugs is determined and used as a basal value. This value issubtracted from the specific binding observed in the presence of addeddrugs. Thus, all data are expressed as the actual amount of bindingabove or below the basal level. Negative values indicate an inhibitionof ³ H!TCP binding relative to this value.

                  TABLE II    ______________________________________                        Enhancement of                         .sup.3 H!TCP Binding               Displacement of         % maximal                .sup.3 H!Glycine Binding                                       glycine    Compound   IC.sub.50, μM                              EC.sub.50, μM                                       response    ______________________________________    2-amino-N-(thieno-               21.8           14.4     39     2,3-b!pyridin-3-yl)-    acetamide    (Reference)    Glycine    0.13           0.056    100    D-cycloserine               2.5            1.8      80    ______________________________________

References:

1. J. W. Olney, Annu. Rev. Pharmacol. Toxicol., 30, 47-71 (1990).

2. B. A. Lawlor and K. L. Davis, Biol. Psychiatry, 31, 337-350 (1992).

3. P. Riederer et al., Arzneim.-Forsch., 42, 265-268 (1992).

4. P. T. Francis et al., Annals New York Academy of Sciences, J. H.Growdon et al., (edits.), 640, 184-188 (1991).

5. L. D. Snell et al., Neuroscience Letters, 83, 313-317 (1987).

6. M. Bradford, Anal. Biochem., 72, 248-254 (1976).

Effective quantities of the compounds of the invention may beadministered to a patient by any of the various methods, for example,orally as in capsules or tablets, parenterally in the form of sterilesolutions or suspensions, and in some cases intravenously in the form ofsterile solutions. The free base final products, while effectivethemselves, may be formulated and administered in the form of theirpharmaceutically acceptable acid addition salts for purposes ofstability, convenience of crystallization, increased solubility and thelike.

Acids useful for preparing the pharmaceutically acceptable acid additionsalts of the invention include inorganic acids such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric and perchloric acids, as wellas organic acids such as tartaric, citric, acetic, succinic, maleic,fumaric and oxalic acids.

The active compounds of the present invention may be orallyadministered, for example, with an inert diluent or with an ediblecarrier, or they may be enclosed in gelatin capsules, or they may becompressed into tablets. For the purpose of oral therapeuticadministration, the active compounds of the invention may beincorporated with excipients and used in the form of tablets, troches,capsules, elixirs, suspensions, syrups, wafers, chewing gum and thelike. These preparations should contain at least 0.5% of activecompounds, but may be varied depending upon the particular form and mayconveniently be between 4% to about 70% of the weight of the unit. Theamount of active compound in such compositions is such that a suitabledosage will be obtained. Preferred compositions and preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between 1.0-300 milligrams of active compound.

The tablets, pills, capsules, troches and the like may also contain thefollowing ingredients: a binder such as micro-crystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, cornstarch and thelike; a lubricant such as magnesium stearate or Sterotex; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin may be added or a flavoring agent such as peppermint, methylsalicylate, or orange flavoring. When the dosage unit form is a capsule,it may contain, in addition to materials of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus, tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes, coloring and flavors. Materials used inpreparing these various compositions should be pharmaceutically pure andnon-toxic in the amounts used.

For the purpose of parenteral therapeutic administration, the activecompounds of the invention may be incorporated into a solution orsuspension. These preparations should contain at least 0.1% of activecompound, but may be varied between 0.5 and about 30% of the weightthereof. The amount of active compound in such compositions is such thata suitable dosage will be obtained. Preferred compositions andpreparations according to the present inventions are prepared so that aparenteral dosage unit contains between 0.5 to 100 milligrams of activecompound.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in disposable syringes or multiple dose vials made ofglass or plastic.

The following examples will further illustrate this invention but arenot intended to limit it in any way. Following Table III specificillustrative preparations of compounds of the invention are described.

                  TABLE III    ______________________________________     ##STR22##    Ex.    No.  A     B     R.sup.1        R.sup.2                                           R.sup.3  m.p. °C.    ______________________________________    1    CH    N     H              4-pyridyl                                           H206-207    2    CH    N     CH.sub.3       4-pyridyl                                           H150-152    3    CH    N     CH.sub.2 CH.sub.3                                    4-pyridyl                                           H123-125    4    CH    N     CH.sub.2 CH.sub.2 CH.sub.3                                    4-pyridyl                                           H256-258    5    CH    N     CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.3                                    4-pyridyl                                           H248-250    6    CH    N     C(O)CH.sub.2 NHCO.sub.2 C(CH.sub.3).sub.3                                    H      H194-196    7    CH    N     C(O)CH.sub.2 NH.sub.2                                    H      H128-129    8    N     CH    H              4-pyridyl                                           H236-238    9    N     CH    CH.sub.3       4-pyridyl                                           H    10   N     CH    CH.sub.2 CH.sub.3                                    4-pyridyl                                           H    11   N     CH    CH.sub.2 CH.sub.2 CH.sub.3                                    4-pyridyl                                           H119-120    12   N     CH    CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.3                                    4-pyridyl                                           H    13   N     CH    C(O)CH.sub.2 NHCO.sub.2 C(CH.sub.3).sub.3                                    H      H    14   N     CH    C(O)CH.sub.2 NH.sub.2                                    H      H    ______________________________________

EXAMPLE 1 3-(4-Pyridinylamino)thieno 2,3-b!pyridine

A solution of 3-aminothieno 2,3-b!pyridine¹,2 (9 g) and 4-chloropyridinehydrochloride (9 g) in 75 mL of 1-methyl-2-pyrrolidinone was stirred at90° C. for one hour. After cooling, the reaction mixture was stirredwith water, washed with ether and separated. The aqueous layer wasbasified with 30% aqueous ammonium hydroxide and extracted with ethylacetate. The organic extract was washed with water and saturated sodiumchloride, and then was dried (anhydrous magnesium sulfate), filtered andevaporated. Elution of the residue through silica with 10% methanol inethyl acetate via flash column chromatography afforded 6 g of a solid,m.p. 200°-204° C. Recrystallization of 2.5 g from acetonitrile afforded2 g of a solid, m.p. 206°-207° C.

¹ L. H. Klemm, et al., J. Heterocyclic Chem., 14, 299 (1977).

² A. D. Dunn and R. Norrie, J. Heterocyclic Chem., 24, 85 (1987).

ANALYSIS:

Calculated for C₁₂ H₉ N₃ S: 63.41% C 3.99% H 18.49% N

Found: 63.09% C 3.86% H 18.75% N

EXAMPLE 2 3-(Methyl-4-pyridinylamino)thieno 2,3-b!pyridine

A solution of 3-(4-pyridinylamino)thieno 2,3-b!pyridine (4 g) in 25 mLof dimethylformamide was added to an ice-cooled suspension of sodiumhydride (60% oil dispersion, 0.85 g, washed with heptane) in 5 mL ofdimethylformamide. After anion formation was completed a solution ofdimethyl sulfate (2.4 g) in 5 mL of dimethylformamide was added. Afterone hour the reaction mixture was poured into ice-water and extractedwith ethyl acetate. The organic extract was washed with water andsaturated sodium chloride, and then was dried (anhydrous magnesiumsulfate), filtered and evaporated to 4 g of a solid. Elution throughsilica with 10% methanol in ethyl acetate via flash columnchromatography afforded 3 g of a solid, m.p. 143°-145° C.Recrystallization from acetonitrile afforded 2.1 g of crystals, m.p.150°-152° C.

ANALYSIS:

Calculated for C₁₃ H₁₁ N₃ S: 64.71% C 4.59% H 17.42% N

Found: 64.47% C 4.55% H 17.39% N

EXAMPLE 3 3-(Ethyl-4-pyridinylamino)thieno 2,3-b!pyridine

A solution of 3-(4-pyridinylamino)thieno 2,3-b!pyridine (4 g) in 25 mLof dimethylformamide was added to an ice-cooled suspension of sodiumhydride (60% oil dispersion, 0.8 g, washed with heptane) in 5 mL ofdimethylformamide. After anion formation was completed a solution ofdiethyl sulfate (3 g) in 5 mL of dimethylformamide was added. After onehour the reaction mixture was poured into ice-water and extracted withethyl acetate. The organic extract was washed with water and saturatedsodium chloride, and then was dried (anhydrous magnesium sulfate),filtered and evaporated to 5 g of an oil. Elution through silica with10% methanol in ethyl acetate via flash column chromatography afforded2.1 g of a solid. Recrystallization from acetonitrile afforded 1.4 g ofcrystals, m.p. 123°-125° C.

ANALYSIS:

Calculated for C₁₄ H₁₃ N₃ S: 65.85% C 5.13% H 16.46% N

Found: 65.56% C 5.14% H 16.57% N

EXAMPLE 4 3-(Propyl-4-pyridinylamino)thieno 2,3-b!pyridine hydrochloride

3-(4-Pyridinylamino)thieno 2,3-b!pyridine (3 g) was added portionwise asa solid to an ice-cooled suspension of sodium hydride (60% oildispersion, 0.8 g, washed with heptane) in 25 mL of dimethylformamide.After anion formation was completed 1-bromopropane (1.9 g) was added.After warming and stirring one hour at ambient temperature the reactionmixture was poured into ice-water and extracted with ether. The organicextract was washed with water and saturated sodium chloride then wasdried (anhydrous magnesium sulfate), filtered and evaporated to 4 g ofan oil. Gradient elution through silica with ethyl acetate followed by10% methanol in ethyl acetate via flash column chromatography afforded3.1 g of a solid, m.p. 136°-137° C. Conversion to the hydrochloride saltin 50% methanol in ether afforded 3 g of a hygroscopic solid.Recrystallization from 5% methanol in ether afforded 2.3 g of a powder,m.p. 256°-258° C.

ANALYSIS:

Calculated for C₁₅ H₁₆ CIN₃ S: 58.91% C 5.27% H 13.74% N

Found: 58.69% C 5.26% H 13.61% N

EXAMPLE 5 3-(Butyl-4-pyridinylamino)thieno 2,3-b!pyridine hydrochloride

A solution of 3-(4-pyridinylamino)thieno 2,3-b!pyridine (4 g) in 25 mLof dimethylformamide was added to an ice-cooled suspension of sodiumhydride (60% oil dispersion, 0.8 g, washed with heptane) in 5 mL ofdimethylformamide. After anion formation was completed a solution of1-bromobutane (2.7 g) in 5 mL of dimethylformamide was added. After onehour the reaction mixture was poured into ice-water and extracted withethyl acetate. The organic extract was washed with water and saturatedsodium chloride, and then was dried (anhydrous magnesium sulfate),filtered and evaporated. Elution through silica with 10% methanol inethyl acetate via flash column chromatography afforded 4.2 g of an oil.Conversion to the hydrochloride salt in 10% methanol in ether afforded3.8 g of a powder, m.p. 248°-250° C.

ANALYSIS:

Calculated for C₁₆ H₁₈ CIN₃ S: 60.08% C 5.67% H 13.14% N

Found: 60.02% C 5.49% H 13.00% N

EXAMPLE 6 tert-Butyl 2-(thieno2,3-b!pyridin-3-ylamino)-2-oxoethyl!carbamate

1,3-Dicyclohexylcarbodiimide (11 g) was added with stirring to asolution of 3-aminothieno 2,3-b!pyridine (8 g) andN-(tert-butoxycarbonyl)glycine (10 g) in 200 mL of dichloromethane.After one hour the reaction mixture was filtered to remove the separated1,3-dicyclohexylurea and evaporated to an oil. Crystallization fromethyl ether afforded 14 g of a solid, m.p. 190°-192° C.Recrystallization of 3 g from acetonitrile afforded 2.4 g of crystals,m.p. 192°-194° C. Final recrystallization from acetonitrile afforded 2.0g of crystals, m.p. 194°-196° C.

ANALYSIS:

Calculated for C₁₄ H₁₇ N₃ O₃ S: 54.71% C 5.57% H 13.67% N

Found: 55.14% C 5.84% H 13.55% N

EXAMPLE 7 2-Amino-N-(thieno 2,3-b!pyridin-3-yl)acetamide

A solution of tert-butyl 2-(thieno 2,3-b!pyridin-3-ylamino)-2-oxoethyl!carbamate (11 g) in 800 mL of methanol and 25 mL of saturated etherealhydrogen chloride was allowed to stand at ambient temperature for twentyhours, and then was evaporated. The residue was dissolved in water,basified with 30% aqueous ammonium hydroxide and extracted with ethylacetate. The organic extract was washed with water and saturated sodiumchloride, and then was dried (anhydrous magnesium sulfate), filtered andevaporated. Gradient elution of the residue through silica with ethylacetate and then with 20% methanol in ethyl acetate via flash columnchromatography afforded 3 g of unreacted carbamate followed by 3 g ofproduct. Trituration with ether afforded 2.5 g of a solid, m.p.138°-139° C. Recrystallization from 10% acetonitrile in ether afforded1.5 g of a light tan solid, m.p. 128°-129° C.

ANALYSIS:

Calculated for C₉ H₉ N₃ OS: 52.15% C 4.38% H 20.28% N

Found: 51.94% C 4.32% H 20.19% N

EXAMPLE 8 3-(4-Pyridinylamino)thieno 2,3-c!pyridine

A solution of 3-aminothieno 2,3-c!pyridine (10 g) and 4-chloropyridinehydrochloride (10 g) in 200 mL of 1-methyl-2-pyrrolidinone was stirredat 80° C. for four hours, and then was cooled, stirred with ice-water,basified with sodium carbonate and extracted with ethyl acetate. Theorganic extract was washed with water and saturated sodium chloride, andthen was dried (an hydrous magnesium sulfate), filtered and evaporated.Gradient elution through silica with dichloromethane followed by 10%methanol in dichloromethane afforded 4 g (26%) of a tan solid, m.p.236°-238° C.

EXAMPLE 11 3-(Propyl-4-pyridinylamino)thieno 2,3-c!pyridine

3-(4-Pyridinylamino)thieno 2,3-c!pyridine (4 g) was added portionwise asa powder to a suspension of sodium hydride (60% oil dispersion, 0.8 gwashed with heptane) in 50 mL of dimethylformamide. After anionformation was completed 1-bromopropane (2.2 g) was added. After stirringone hour the reaction mixture was poured into ice-water and extractedwith ethyl acetate. The organic extract was washed with water saturatedsodium chloride, and then was dried (anhydrous magnesium sulfate),filtered and evaporated to 5 g of a dark oil. Elution through silicawith 5% methanol in ethyl acetate via flash column chromatographyafforded 3.5 g of a yellow solid. Elution through alumina with ether viacolumn chromatography afforded 2.8 g of a yellow solid.Recrystallization from heptane afforded 2.6 g (54.8%) of yellowishcrystals, m.p. 119°-120° C.

ANALYSIS:

Calculated for C₁₅ H₁₅ N₃ S: 66.88% C 5.61% H 15.60% N

Found: 66.78% C 5.56% H 15.52% N

It should be understood that this specification and examples are setforth by way of illustration and not limitation and that variousmodifications and changes may be made without departing from the spiritand scope of the present invention as defined by the appended claims.

We claim:
 1. A compound of the formula ##STR23## where R¹ is hydrogen,(C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃ -C₆)alkynyl, aryl(C₁ -C₆)alkyl, (C₁-C₆)alkylcarbonyl, formyl, (C₁ -C₆)alkoxycarbonyl, aryl(C₁-C₆)alkoxycarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl(C₁ -C₆)alkylamino(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₁₈)alkylcarbonyl,(C₁ -C₆)dialkylamino(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl, (C₁-C₆)alkylamino(C₁ -C₆)alkyl, or (C₁ -C₆)dialkylamino(C₁ -C₆)alkyl;R² ishydrogen or (C₁ -C₆)alkyl; with the proviso that R¹ andR² are notconcurrently hydrogen; and R³ is hydrogen, (C₁ -C₆)alkyl or (C₁-C₆)alkoxycarbonyl;n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; with theproviso that the sum of m and n is always 3; and pharmaceuticallyacceptable addition salts thereof and optical or geometrical isomers orracemic mixtures thereof.
 2. The compound of claim 1 of the formula##STR24## where R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃-C₆)alkynyl, aryl(C₁ -C₆)alkyl, aryl(C₁ -C₆)alkoxycarbonyl, (C₁-C₆)alkylcarbonyl, formyl, (C₁ -C₆)alkoxycarbonyl, (C₁-C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl, aryl(C₁-C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl, (C₁ -C₆)alkylamino(C₁-C₆)alkylcarbonyl, amino(C₁ -C₁₈)alkylcarbonyl, (C₁ -C₆)dialkylamino(C₁-C₆)alkylcarbonyl, amino(C₁ -C₆)alkyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkyl,or (C₁ -C₆)dialkylamino(C₁ -C₆)alkyl;R² is hydrogen or (C₁ -C₆)alkyl;with the proviso that R¹ and R² are not concurrently hydrogen; and R³ ishydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl;pharmaceuticallyacceptable addition salts thereof and optical or geometrical isomers orracemic mixtures thereof.
 3. The compound of claim 2 of the formula##STR25## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl or amino(C₁ -C₁₈)alkylcarbonyl; andR³ is hydrogen. 4.The compound of claim 3 wherein R¹ is (C₁-C₆)alkoxycarbonylamino(C₁)alkylcarbonyl or amino(C₁)alkylcarbonyl. 5.The compound of claim 4 which is 2-amino-N-(thieno2,3-b!pyridin-3-yl)acetamide.
 6. The compound of claim 4 which ist-butyl 2-(thieno 2,3-b!pyridin-3-ylamino)-2-oxoethyl!carbamate.
 7. Thecompound of claim 1 of the formula ##STR26## wherein R¹ is hydrogen, (C₁-C₆) alkyl, (C₃ -C₆)alkenyl, (C₃ -C₆)alkynyl, aryl(C₁ -C₆)alkyl, aryl(C₁-C₆)alkoxycarbonyl, (C₁ -C₆)alkylcarbonyl, formyl, (C₁-C₆)alkoxycarbonyl, (C₁ -C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl,aryl(C₁ -C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl, (C₁-C₆)alkylamino-(C₁ -C₆)alkylcarbonyl, amino(C₁ -C₁₈)alkylcarbonyl, (C₁-C₆)dialkylamino-(C₁ -C₆)alkylcarbonyl, (C₁ -C₆)alkoxycarbonyl, amino(C₁-C₆)alkyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkyl, or (C₁ -C₆)dialkylamino(C₁-C₆)alkyl;R² is hydrogen or (C₁ -C₆)alkyl; with the proviso that R¹ andR² are not concurrently hydrogen; and R³ is hydrogen, (C₁ -C₆)alkyl or(C₁ -C₆)alkoxycarbonyl; and pharmaceutically acceptable acid additionsalts thereof and optical or geometrical isomers or racemic mixturesthereof.
 8. The compound of claim 1 of the formula ##STR27## wherein R¹is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃ -C₆)alkynyl, aryl(C₁-C₆)alkyl, aryl(C₁ -C₆)alkoxycarbonyl, (C₁ -C₆)alkylcarbonyl, formyl,(C₁ -C₆)alkoxycarbonyl, (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkylcarbonyl, amino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)dialkylamino(C₁ -C₆)alkylcarbonyl, amino(C₁-C₆)alkyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkyl, or (C₁ -C₆)dialkylamino(C₁-C₆)alkyl;R² is hydrogen or (C₁ -C₆)alkyl; with the proviso that R¹ andR² are not concurrently hydrogen; and R³ is hydrogen, (C₁ -C₆)alkyl or(C₁ -C₆)alkyl or (C₁ -C₆)alkoxycarbonyl; and pharmaceutically acceptableaddition salts thereof and optical or geometrical isomers or racemicmixtures thereof.
 9. The compound of claim 1 of the formula ##STR28##wherein R¹ is hydrogen, (C₁ -C₆)alkyl, (C₃ -C₆)alkenyl, (C₃ -C₆)alkynyl,aryl(C₁ -C₆)alkyl, aryl(C₁ -C₆)alkoxycarbonyl, (C₁ -C₆)alkylcarbonyl,formyl, (C₁ -C₆)alkoxycarbonyl, (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkylcarbonyl, amino(C₁-C₁₈)alkylcarbonyl, (C₁ -C₆)dialkylamino(C₁ -C₆)alkylcarbonyl, amino(C₁-C₆)alkyl, (C₁ -C₆)alkylamino(C₁ -C₆)alkyl, or (C₁ -C₆)dialkylamino(C₁-C₆)alkyl;R² is hydrogen or (C₁ -C₆)alkyl; with the proviso that R¹ andR² are not concurrently hydrogen; and R³ is hydrogen, (C₁ -C₆)alkyl or(C₁ -C₆)alkoxycarbonyl; and pharmaceutically acceptable salts thereofand optical or geometrical isomers or racemic mixtures thereof.
 10. Thecompound of claim 7 of the formula ##STR29## wherein R¹ is (C₁-C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl, aryl(C₁-C₆)alkoxycarbonylamino(C₁ -C₁₈)alkylcarbonyl or amino(C₁-C₁₈)alkylcarbonyl; andR³ is hydrogen.
 11. The compound of claim 10wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁)alkylcarbonyl oramino(C₁)alkylcarbonyl.
 12. The compound of claim 9 of the formula##STR30## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl or amino(C₁ -C₆)alkylcarbonyl; andR³ is hydrogen. 13.The compound of claim 12 wherein R¹ is (C₁-C₆)alkoxycarbonylamino(C₁)alkylcarbonyl or amino(C₁)alkylcarbonyl. 14.A pharmaceutical composition which comprises a glycine partial agonisteffective amount of the compound of claim 3 and a pharmaceuticallyacceptable carrier.
 15. A pharmaceutical composition which comprises aglycine partial agonist effective amount of the compound of claim 10 anda pharmaceutically acceptable carrier.
 16. A pharmaceutical compositionwhich comprises a glycine partial agonist effective amount of thecompound of claim 12 and a pharmaceutically acceptable carrier.
 17. Apharmaceutical composition which comprises a glycine partial agonisteffective amount of the compound of claim 4 and a pharmaceuticallyacceptable carrier.
 18. A method for modulating the binding of glycineassociated with the N-methyl-D-aspartate receptor complex whichcomprises administering the compound of claim 3 in an amount effectiveto interact with the glycine binding site of the N-methyl-D-aspartatereceptor complex.
 19. A method for modulating the binding of glycineassociated with the N-methyl-D-aspartate receptor complex whichcomprises administering the compound of claim 10 in an amount effectiveto interact with the glycine binding site of the N-methyl-D-aspartatereceptor complex.
 20. A method for modulating the binding of glycineassociated with the N-methyl-D-aspartate receptor complex whichcomprises administering the compound of claim 4 in an amount effectiveto interact with the glycine binding site of the N-methyl-D-aspartatereceptor complex.
 21. A method for modulating the binding of glycineassociated with the N-methyl-D-aspartate receptor complex whichcomprises administering the compound of claim 12 in an amount effectiveto interact with the glycine binding site of the N-methyl-D-aspartatereceptor complex.
 22. A method of treating a condition which isameliorated by the use of a glycine partial agonist which comprisesadministering to a patient an effective amount to relieve the conditionof the compound of claim
 3. 23. A method of treating a condition whichis ameliorated by the use of a glycine partial agonist which comprisesadministering to a patient an effective amount to relieve the conditionof the compound of claim
 10. 24. The compound of claim 8 of the formula##STR31## wherein R¹ is (C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl, aryl(C₁ -C₆)alkoxycarbonylamino(C₁-C₁₈)alkylcarbonyl or amino(C₁ -C₆)alkylcarbonyl; andR³ is hydrogen. 25.The compound of claim 24 wherein R¹ is (C₁-C₆)alkoxycarbonylamino(C₁)alkylcarbonyl or amino(C₁)alkylcarbonyl.